Related Experiment Video
Updated: Jun 21, 2026
Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy
Published on: November 4, 2018
Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci
Zongqiang Cui1, Ke Zhang, Zhiping Zhang
1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Abstract:
The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14-22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.
Insights
Human Cytomegalovirus protein pp65 dynamically forms nuclear foci and self-associates via its N-terminus. The ppUL97 phosphorylation site, not the self-association domain, dictates pp65 localization in these foci.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Human Cytomegalovirus (HCMV) protein pp65 is a major tegument component crucial for infection and assembly.
- Understanding pp65's molecular interactions and localization is key to elucidating HCMV pathogenesis.
Purpose of the Study:
- To investigate the self-interaction and intracellular localization mechanisms of HCMV pp65.
- To identify the domains responsible for pp65 self-association and nuclear foci localization.
Main Methods:
- Live cell imaging and Fluorescence Recovery After Photobleaching (FRAP) to observe pp65 dynamics.
- Fluorescence Resonance Energy Transfer (FRET) microscopy to study protein-protein interactions.
- Yeast two-hybrid assays to confirm self-association.
Main Results:
- HCMV pp65 dynamically accumulates in punctate nuclear foci in mammalian cells.
- pp65 exhibits self-interaction, mediated by N-terminal amino acids 14-22.
- The ppUL97 phosphorylation site, distinct from the self-association domain, is essential for pp65 localization to nuclear foci.
Conclusions:
- pp65 self-association and nuclear foci localization are distinct processes mediated by different protein regions.
- The interaction with ppUL97 and its phosphorylation site are critical for pp65's specific nuclear localization during HCMV infection.
- These findings provide insights into HCMV assembly and infection mechanisms.
